Crystalline salts of a plasma kallikrein inhibitor

ABSTRACT

Disclosed are crystalline salts of Compound I, methods of preparing them, and related pharmaceutical preparations thereof. Also disclosed are methods of treatment using the crystalline salts of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/671,649, filedNov. 1, 2019; which claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/754,983, filed Nov. 2, 2018.

BACKGROUND

Serine proteases make up the largest and most extensively studied groupof proteolytic enzymes. Their critical roles in physiological processesextend over such diverse areas as blood coagulation, fibrinolysis,complement activation, reproduction, digestion, and the release ofphysiologically active peptides. Many of these vital processes beginwith cleavage of a single peptide bond or a few peptide bonds inprecursor protein or peptides.

Sequential limited proteolytic reactions or cascades are involved inblood clotting, fibrinolysis, and complement activation. The biologicalsignals to start these cascades can be controlled and amplified as well.Similarly, controlled proteolysis can shut down or inactivate proteinsor peptides through single bond cleavages.

Kallikreins are a subgroup of serine proteases. In humans, plasmakallikrein (KLKB 1) has no known homologue, while tissuekallikrein-related peptidases (KLKs) encode a family of fifteen closelyrelated serine proteases. Plasma kallikrein participates in a number ofpathways relating to the intrinsic pathway of coagulation, inflammation,and the complement system.

Coagulation is the process by which blood forms clots, for example tostop bleeding. The physiology of coagulation is somewhat complex insofaras it includes two separate initial pathways, which converge into afinal common pathway leading to clot formation. In the final commonpathway, prothrombin is converted into thrombin, which in turn convertsfibrinogen into fibrin, the latter being the principal building block ofcross-linked fibrin polymers which form a hemostatic plug. Of the twoinitial pathways upstream of the final common pathway, one is known asthe contact activation or intrinsic pathway, and the other is known asthe tissue factor or extrinsic pathway.

The intrinsic pathway begins with formation of a primary complex oncollagen by high-molecular-weight kininogen (HMWK), prekallikrein, andFXII (Factor XII; Hageman factor). Prekallikrein is converted tokallikrein, and FXII is activated to become FXIIa. FXIIa then convertsFactor XI (FXI) into FXIa, and FXIa in turn activates Factor IX (FIX),which with its co-factor FVIIIa form the “tenase” complex, whichactivates Factor X (FX) to FXa. It is FXa which is responsible for theconversion of prothrombin into thrombin within the final common pathway.

Prekallikrein, the inactive precursor of plasma kallikrein, issynthesized in the liver and circulates in the plasma bound to HMWK oras a free zymogen. Prekallikrein is cleaved by activated factor XII(FXIIa) to release activated plasma kallikrein (PK). Activated plasmakallikrein displays endopeptidase activity towards peptide bonds afterarginine (preferred) and lysine. PK then generates additional FXIIa in afeedback loop which in turn activates factor XI (FXI) to FXIa to connectto the common pathway. Although the initial activation of the intrinsicpathway is through a small amount of FXIIa activating a small amount ofPK, it is the subsequent feedback activation of FXII by PK that controlsthe extent of activation of the intrinsic pathway and hence downstreamcoagulation. Hathaway, W. E., et al. (1965) Blood 26:521-32.

Activated plasma kallikrein also cleaves HMWK to release the potentvasodilator peptide bradykinin. It is also able to cleave a number ofinactive precursor proteins to generate active products, such as plasmin(from plasminogen) and urokinase (from prourokinase). Plasmin, aregulator of coagulation, proteolytically cleaves fibrin into fibrindegradation products that inhibit excessive fibrin formation.

Patients who have suffered acute myocardial infarction (MI) showclinical evidence of being in a hypercoagulable (clot-promoting) state.This hypercoagulability is paradoxically additionally aggravated inthose receiving fibrinolytic therapy. Increased generation of thrombin,as measured by thrombin-antithrombin III (TAT) levels, is observed inpatients undergoing such treatment compared to the already high levelsobserved in those receiving heparin alone. Hoffmeister, H. M. et al.(1998) Circulation 98:2527-33. The increase in thrombin has beenproposed to result from plasmin-mediated activation of the intrinsicpathway by direct activation of FXII by plasmin.

Not only does the fibrinolysis-induced hypercoagulability lead toincreased rates of reocclusion, but it is also probably responsible, atleast in part, for failure to achieve complete fibrinolysis of the clot(thrombus), a major shortcoming of fibrinolytic therapy (Keeley, E. C.et al. (2003) Lancet 361: 13-20). Another problem in fibrinolytictherapy is the accompanying elevated risk of intracranial hemorrhage.Menon, V. et al. (2004) (Chest 126:549S-575S; Fibrinolytic TherapyTrialists' Collaborative Group (1994) Lancet 343:311-22. Hence, anadjunctive anti-coagulant therapy that does not increase the risk ofbleeding, but inhibits the formation of new thrombin, would be greatlybeneficial.

Plasma kallikrein inhibitors also have therapeutic potential fortreating hereditary angioedema (HAE). HAE is a serious and potentiallylife-threatening rare genetic illness, caused by mutations in theC1-esterase inhibitor (C1INH) gene, located on chromosome 11q. HAE isinherited as an autosomal dominant condition, although one quarter ofdiagnosed cases arise from a new mutation. HAE has been classed as anorphan disease in Europe, with an estimated prevalence of 1 in 50,000.Individuals with HAE experience recurrent acute attacks of painfulsubcutaneous or submucosal edema of the face, larynx, gastrointestinaltract, limbs or genitalia which, if untreated, may last up to 5 days.Attacks vary in frequency, severity and location and can belife-threatening. Laryngeal attacks, with the potential forasphyxiation, pose the greatest risk. Abdominal attacks are especiallypainful, and often result in exploratory procedures or unnecessarysurgery. Facial and peripheral attacks are disfiguring and debilitating.

HAE has a number of subtypes. HAE type I is defined by C1INH genemutations which produce low levels of C1-inhibitor, whereas HAE type IIis defined by mutations which produce normal levels of ineffective C1protein. HAE type III has separate pathogenesis, being caused bymutations in the F12 gene which codes for the serine protease known asFactor XII. Diagnostic criteria for distinguishing the subtypes of HAE,and distinguishing HAE from other angioedemas, can be found in AnnAllergy Asthma Immunol 2008; 100(Suppl2): S30-S40 and J Allergy ClinImmunol 2004; 114: 629-37, incorporated herein by reference.

Current treatments for HAE fall into two main types. Older non-specifictreatments including androgens and antifibrinolytics are associated withsignificant side effects, particularly in females. Newer treatments arebased on an understanding of the molecular pathology of the disease,namely that C1INH is the most important inhibitor of kallikrein in humanplasma and that C INH deficiency leads to unopposed activation of thekallikrein-bradykinin cascade, with bradykinin the most importantmediator of the locally increased vascular permeability that is thehallmark of an attack. All of the currently available targeted therapiesare administered by intravenous or subcutaneous injection. There iscurrently no specific targeted oral chronic therapy for HAE.

Therefore, a need exists to develop inhibitors of PK that can tip thebalance of fibrinolysis/thrombosis at the occluding thrombus towarddissolution, thereby promoting reperfusion and also attenuating thehypercoagulable state, thus preventing thrombus from reforming andreoccluding the vessel. In particular, the creation of plasma kallikreininhibitors that are specific and capable of being formulated for in vivouse could lead to a new class of therapeutics. Thus, what is needed areimproved compositions and methods for preparing and formulating plasmakallikrein inhibitors.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a crystalline salt of Compound I,

Another aspect of the invention relates to methods for preparing thecrystalline salts of Compound I. In such aspects, the methods comprisea) providing a freebase mixture of Compound I in a first organicsolvent; b) combining the freebase mixture with a reagent solutioncomprising an acid and a second organic solvent under conditionssufficient to form a mixture comprising a salt of Compound I; and c)crystallizing the salt of Compound I from the mixture comprising a saltof Compound I.

In certain embodiments, the present invention provides a pharmaceuticalcomposition, comprising a crystalline salt of Compound I, and one ormore pharmaceutically acceptable excipients. In certain embodiments, thepharmaceutical preparations may be for use in treating or preventing acondition or disease characterized by aberrant plasma kallikreinactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray powder diffraction (XRPD) pattern of CompoundI•2(HCl).

FIG. 2 is a spectrum showing a TG-IR analysis of Compound I•2(HCl).

FIG. 3 shows plasma concentration of Compound I•2(HCl) versus timeprofiles on Day 7 after once per day (QD) oral administration ofCompound I•2(HCl) at 125, 250, and 500 mg QD; and on Day 14 after QDoral administration of Compound I•2(HCl) at 350 mg QD.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, the invention provides a crystalline salt ofCompound I,

Compound I is a potent inhibitor of plasma kallikrein, described in WO2015/134998 and U.S. Patent Application Publication No. 2017/0073314 A1,the contents of both of which are hereby incorporated by reference.

A crystalline form of Compound I can be used to modulate/improve thephysicochemical properties of the compound, including but not limited tosolid state properties (e.g., crystallinity, hygroscopicity, meltingpoint, or hydration), pharmaceutical properties (e.g.,solubility/dissolution rate, stability, or compatibility), as well ascrystallization characteristics (e.g., purity, yield, or morphology).For example, crystallization of Compound I enables access to thecompound with consistent and predictable purity levels. Additionally,the uniform particle sizes resulting from crystallization lead toimproved processability of the solid crystalline compound relative tothe solid amorphous form. The crystalline form of Compound I exhibitsbeneficial pharmacokinetic properties as well; the uniformity of thecrystal form leads to a consistent and predictable pharmacokineticprofile.

In certain embodiments, the crystalline salt is a hydrochloride salt,e.g., a bis(hydrochloride) salt.

In certain embodiments, the polymorph of the crystalline salt ischaracterized by X-ray powder diffraction (XRPD). θ represents thediffraction angle, measured in degrees. In certain embodiments, thediffractometer used in XRPD measures the diffraction angle as two timesthe diffraction angle θ. Thus, in certain embodiments, the diffractionpatterns described herein refer to X-ray intensity measured againstangle 2θ.

The peak value at the diffraction angle θ, or at two times thediffraction angle θ (2θ), may exhibit a minor measurement error due tothe XRPD instruments or the conditions under which the measurement istaken. Accordingly, in certain embodiments, the characteristic peaks inthe XRPD pattern have a value of ° 2θ±0.2°. In certain embodiments, thecharacteristic peaks in the XRPD pattern have a value of ° 2θ±0.10. Incertain embodiments, the characteristic peaks in the XRPD pattern have avalue of ° 2θ±0.06°.

In certain embodiments, the crystalline salt of Compound I hascharacteristic peaks in the X-ray powder diffraction (XRPD) pattern atvalues of two theta (° 2θ±0.2°) of 5.3, 9.0, and 22.0. In certainembodiments, the crystalline salt of Compound I has characteristic peaksin the X-ray powder diffraction (XRPD) pattern at values of two theta (°2θ) of 5.3, 9.0, and 22.0.

In further embodiments, the crystalline salt has characteristic peaks inthe X-ray powder diffraction (XRPD) pattern at values of two theta (°2θ±0.2°) of 5.3, 9.0, 19.8, 21.2, 22.0, and 23.3. In certainembodiments, the crystalline salt has characteristic peaks in the X-raypowder diffraction (XRPD) pattern at values of two theta (° 2θ) of 5.3,9.0, 19.8, 21.2, 22.0, and 23.3.

In yet further embodiments, the crystalline salt has characteristicpeaks in the X-ray powder diffraction (XRPD) pattern at values of twotheta (° 2θ±0.2°) of 5.3, 9.0, 14.3, 16.2, 19.8, 21.2, 22.0, 23.3, 24.6,and 30.3. In certain embodiments, the crystalline salt hascharacteristic peaks in the X-ray powder diffraction (XRPD) pattern atvalues of two theta (° 2θ) of 5.3, 9.0, 14.3, 16.2, 19.8, 21.2, 22.0,23.3, 24.6, and 30.3.

In certain embodiments, the crystalline salt of Compound I hascharacteristic peaks in the X-ray powder diffraction (XRPD) pattern atvalues of two theta (° 2θ±0.2°) of 5.28, 8.96, and 22.01. In certainembodiments, the crystalline salt of Compound I has characteristic peaksin the X-ray powder diffraction (XRPD) pattern at values of two theta (°2θ) of 5.28, 8.96, and 22.01.

In further embodiments, the crystalline salt has characteristic peaks inthe X-ray powder diffraction (XRPD) pattern at values of two theta (°2θ±0.2°) of 5.28, 8.96, 19.79, 21.16, 22.01, and 23.31. In certainembodiments, the crystalline salt of Compound I has characteristic peaksin the X-ray powder diffraction (XRPD) pattern at values of two theta (°2θ) of 5.28, 8.96, 19.79, 21.16, 22.01, and 23.31.

In further embodiments, the crystalline salt has characteristic peaks inthe X-ray powder diffraction (XRPD) pattern at values of two theta (°2θ±0.2°) of 5.28, 8.96, 14.27, 16.18, 19.79, 21.16, 22.01, 23.31, 24.64,and 30.31. In certain embodiments, the crystalline salt of Compound Ihas characteristic peaks in the X-ray powder diffraction (XRPD) patternat values of two theta (° 2θ) of 5.28, 8.96, 14.27, 16.18, 19.79, 21.16,22.01, 23.31, 24.64, and 30.31.

In certain embodiments, the crystalline salt of Compound I has an XRPDpattern substantially similar to that shown in FIG. 1.

The relative intensity, as well as the two theta value, of each peak inthe XRPD patterns described above, as well as shown in FIG. 1, maychange or shift under certain conditions, although the crystalline formis the same. By comparing XRPD data sets, one of ordinary skill in theart should be able to readily determine whether a given crystalline formis the same crystalline form as described above and shown in FIG. 1.

In certain embodiments, the crystalline salt of Compound I is complexedwith water in a molar ratio of 1:1 crystalline salt to water. In certainsuch embodiments, the crystal lattice does not comprise molecules ofwater.

In certain embodiments, the crystalline salt of Compound I is complexedwith water in a molar ratio of 1:2 crystalline salt to water. In certainsuch embodiments, the crystal lattice does not comprise molecules ofwater.

In certain embodiments, the crystalline salt of Compound I is complexedwith water in a molar ratio of 1:2.5 crystalline salt to water. Incertain such embodiments, the crystal lattice does not comprisemolecules of water.

In certain embodiments, the crystalline salt of Compound I has athermogravimetric-infrared spectrum substantially similar to that shownin FIG. 2.

The term “substantially pure” as used herein, refers to a crystallinepolymorph that is greater than 90% pure, meaning that it contains lessthan 10% of any other compound, including the corresponding amorphouscompound or an alternative polymorph of the crystalline salt.Preferably, the crystalline polymorph is greater than 95% pure, or evengreater than 98% pure.

In certain embodiments, the crystalline salt of Compound I hascharacteristic peaks in the XRPD pattern at values of two theta (° 2θ)as described herein above and is substantially pure. For example, thecrystalline salt form can be at least 90% pure, preferably at least 95%pure, or more preferably at least 98% pure.

In certain embodiments, the crystalline salt of Compound I has an XRPDpattern that is substantially the same as that shown in FIG. 1 and issubstantially pure. For example, the crystalline salt form can be atleast 90% pure, preferably at least 95% pure, or more preferably atleast 98% pure.

Methods of Making the Crystalline Salts

In certain embodiments, the invention relates to a method for thepreparation of a crystalline salt of Compound I, comprising a) providinga freebase mixture of Compound I in a first organic solvent; b)combining the freebase mixture with a reagent solution comprising anacid and a second organic solvent under conditions sufficient to form amixture comprising a salt of Compound I; and c) crystallizing the saltof Compound I from the mixture comprising a salt of Compound I.

In certain embodiments, the crystalline salt is a hydrochloride salt,e.g., a bis(hydrochloride) salt.

In certain embodiments, the first organic solvent comprises a polaraprotic solvent, such as acetonitrile, N,N-dimethylacetamide (DMA),dimethylformamide (DMF), dimethylsulfoxide (DMSO), diethyl ether, ethylacetate, isopropyl acetate, methylethyl ketone, methyl tert-butyl ether(MTBE), N-methyl-2-pyrrolidone (NMP), tetrahydrofuran. Preferably, thepolar aprotic solvent is methyl tert-butyl ether.

In certain embodiments, the first organic solvent further comprises anon-polar solvent. Non-polar solvents include, for example, benzene,heptaine, hexanes, and toluene. In certain embodiments, the non-polarsolvent is toluene.

In certain embodiments, the acid is hydrochloric acid.

In certain embodiments, the second organic solvent is a polar proticsolvent, such as ethanol, methanol, 2-propanol, 1-butanol, water, or anycombination thereof. Preferably, the polar protic solvent is methanol.

In certain embodiments, the mixture comprising a salt of Compound Iformed in step b) is a solution.

In certain embodiments, the mixture comprising the salt of Compound I isa solution, and the step of crystallizing the salt from the mixturecomprises bringing the solution to supersaturation to cause Compound Ito precipitate out of solution.

In certain embodiments, bringing the mixture comprising the salt ofCompound I to supersaturation comprises the slow addition of ananti-solvent, such as heptanes, hexanes, ethanol, or another polar ornon-polar liquid miscible with the organic solvent, allowing thesolution to cool (with or without seeding the solution), reducing thevolume of the solution, or any combination thereof. In certainembodiments, bringing the mixture comprising the salt of Compound I tosupersaturation comprises adding an anti-solvent, cooling the solutionto ambient temperature or lower, and reducing the volume of thesolution, e.g., by evaporating solvent from the solution. In certainembodiments, allowing the solution to cool may be passive (e.g.,allowing the solution to stand at ambient temperature) or active (e.g.,cooling the solution in an ice bath or freezer).

In certain embodiments, the preparation method further comprisesinducing crystallization. The method can also comprise the step ofdrying the crystals, for example under reduced pressure. In certainembodiments, inducing precipitation or crystallization comprisessecondary nucleation, wherein nucleation occurs in the presence of seedcrystals or interactions with the environment (crystallizer walls,stirring impellers, sonication, etc.). In certain embodiments, inducingcrystallization comprises seeding the solution with crystalline seeds ofthe salt of Compound I.

In certain embodiments, the preparation method further comprisesisolating the salt crystals, e.g., by filtering the crystals, bydecanting fluid from the crystals, or by any other suitable separationtechnique. In further embodiments, the preparation method furthercomprises washing the crystals.

In certain embodiments, washing the crystals comprises washing with aliquid selected from anti-solvent, acetonitrile, ethanol, heptanes,hexanes, methanol, tetrahydrofuran, toluene, water, or a combinationthereof. As used herein, “anti-solvent” means a solvent in which thesalt crystals are insoluble, minimally soluble, or partially soluble. Inpractice, the addition of an anti-solvent to a solution in which thesalt crystals are dissolved reduces the solubility of the salt crystalsin solution, thereby stimulating precipitation of the salt. In certainembodiments, the crystals are washed with a combination of anti-solventand the organic solvent. In certain embodiments, the anti-solvent iswater, while in other embodiments it is an alkane solvent, such ashexane or pentane, or an aromatic hydrocarbon solvent, such as benzene,toluene, or xylene.

In certain embodiments, washing the crystals comprises washing thecrystalline salt of Compound I with a solvent or a mixture of one ormore solvents, which are described above. In certain embodiments, thesolvent or mixture of solvents is cooled prior to washing.

In certain embodiments, the method further comprises drying thecrystalline salt.

The crystalline salts described herein may be made according to theabove-described method.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions, each comprising oneor more crystalline salts of the invention and a pharmaceuticallyacceptable carrier. In certain embodiments, the pharmaceuticalcomposition comprises a crystalline salt of the invention and apharmaceutically acceptable carrier. In certain embodiments, thepharmaceutical composition comprises a plurality of crystalline salts ofthe invention and a pharmaceutically acceptable carrier.

The terms “carrier” and “pharmaceutically acceptable carrier” as usedherein refer to a diluent, adjuvant, excipient, or vehicle with which acompound is administered or formulated for administration. Non-limitingexamples of such pharmaceutically acceptable carriers include liquids,such as water, saline, and oils; and solids, such as gum acacia,gelatin, starch paste, talc, keratin, colloidal silica, urea, and thelike. In addition, auxiliary, stabilizing, thickening, lubricating,flavoring, and coloring agents may be used. Other examples of suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences by E. W. Martin, herein incorporated by reference in itsentirety.

In certain embodiments, a pharmaceutical composition of the inventionfurther comprises at least one additional pharmaceutically active agentother than a compound of the invention. The at least one additionalpharmaceutically active agent can be an agent useful in the treatment ofa disease or condition characterized by aberrant plasma kallikreinactivity. For example, the at least one additional pharmaceuticallyactive agent can be an anticoagulation agent, an anti-platelet agent, ora thrombolytic agent.

Anticoagulation agents prevent the coagulation of blood components andthus prevent clot formation, for example in atrial fibrillation.Anticoagulants include, but are not limited to, heparin, warfarin,coumadin, dicumarol, phenprocoumon, acenocoumarol, ethyl biscoumacetate,hirudin, bivalarutin, direct thrombin inhibitors, and indandionederivatives.

Anti-platelet agents inhibit platelet aggregation and are often used toprevent thromboembolic stroke in patients who have experienced atransient ischemic attack, stroke, or atrial fibrillation. Anti-plateletagents include, but are not limited to, aspirin, thienopyridinederivatives such as ticlopodine and clopidogrel, dipyridamole, andsulfinpyrazone, as well as RGD mimetics.

Thrombolytic agents lyse clots that cause thromboembolic phenomena suchas stroke, myocardial infarction, and pulmonary thromboembolism.Thrombolytic agents include, but are not limited to, plasminogen,a2-antiplasmin, streptokinase, antistreplase, TNK, tissue plasminogenactivator (tPA), and urokinase. Tissue plasminogen activator includesnative tPA and recombinant tPA, as well as modified forms of tPA thatretain the enzymatic or fibrinolytic activities of native tPA.

Pharmaceutical compositions of the invention can be prepared bycombining one or more crystalline salts of the invention with apharmaceutically acceptable carrier and, optionally, one or moreadditional pharmaceutically active agents.

In certain embodiments, the invention provides a pharmaceuticalcomposition that is formulated for the prophylactic or therapeutictreatment of a disease or condition characterized by aberrant plasmakallikrein activity.

Methods of Use

The present invention provides compounds that inhibit the formation ofthrombin via the intrinsic pathway and thus reduce the risk of newpathogenic thrombus formation (vessel occlusion or reocclusion) and alsoimprove fibrinolytic-induced reperfusion when given as adjunctivetherapy with a fibrinolytic regimen. Diseases and conditions that can betreated using the compounds of the present invention include, but arenot limited to, stroke, inflammation, reperfusion injury, acutemyocardial infarction, deep vein thrombosis, post fibrinolytic treatmentcondition, angina, edema, angioedema, hereditary angioedema, sepsis,arthritis, hemorrhage, blood loss during cardiopulmonary bypass,inflammatory bowel disease, diabetes mellitus, retinopathy, diabeticretinopathy, diabetic macular edema, diabetic macular degeneration,age-related macular edema, age-related macular degeneration,proliferative retinopathy, neuropathy, hypertension, brain edema,increased albumin excretion, macroalbuminuria, and nephropathy.

For example, in patients with angioedema conditions, small polypeptidePK inhibitor DX-88 (ecallantide) alleviates edema in patients withhereditary angioedema (HAE). Williams, A. et al. (2003) Transfus. Apher.Sci. 29:255-8; Schneider, L. et al. (2007) J Allergy Clin Immunol.120:416-22; and Levy, J. H. et al. (2006) Expert Opin. Invest. Drugs15:1077-90. A bradykinin B2 receptor antagonist, Icatibant, is alsoeffective in treating HAE. Bork, K. et al. (2007) J. Allergy Clin.Immunol. 119:1497-1503. Because plasma kallikrein generates bradykinin,inhibition of plasma kallikrein is expected to inhibit bradykininproduction.

For example, in coagulation resulting from fibrinolytic treatment (e.g.,treatment with tissue plasminogen activator or streptokinase), higherlevels of plasma kallikrein are found in patients undergoingfibrinolysis. Hoffmeister, H. M. et al. (1998) J. Cardiovasc. Pharmacol.31:764-72. Plasmin-mediated activation of the intrinsic pathway has beenshown to occur in plasma and blood and was markedly attenuated in plasmafrom individuals deficient in any of the intrinsic pathway components.Ewald, G. A. et al. (1995) Circulation 91:28-36.

Individuals who have had an acute MI were found to have elevated levelsof activated plasma kallikrein and thrombin. Hoffmeister, H. M., et al.(1998) Circulation 98:2527-33.

DX-88 reduced brain edema, infarct volume, and neurological deficits inan animal model of ischemic stroke. Storini, C. et al. (2006)J. Pharm.Exp. Ther. 318:849-854. C1-inhibitor reduced infarct size in a mousemodel of middle cerebral artery occlusion (MCAO). De Simoni, M. G. etal. (2004) Am. J. Pathol. 164:1857-1863; and Akita, N. et al. (2003)Neurosurgery 52:395-400). B2 receptor antagonists were found to reducethe infarct volume, brain swelling, and neutrophil accumulation and wereneuroprotective in an MCAO animal model. Zausinger, S. et al. (2003)Acta Neurochir. Suppl. 86:205-7; Lumenta, D. B. et al. (2006) Brain Res.1069:227-34; Ding-Zhou, L. et al. (2003) Br. J Pharmacol. 139:1539-47.

Regarding blood loss during cardiopulmonary bypass (CPB), it has beenfound that the kallikrein-kinin (i.e., contact) system is activatedduring CABG. Wachtfogel, Y. T. (1989) Blood 73:468. Activation of thecontact system during CPB results in up to a 20-fold increase in plasmabradykinin. Cugno, M. et al. (2006) Chest 120:1776-82; and Campbell, D.J. et al. (2001) Am. J. Physiol. Reg. Integr. Comp. Physiol.281:1059-70.

Plasma kallikrein inhibitors P8720 and PKSI-527 have also been found toreduce joint swelling in rat models of arthritis. De La Cadena, R. A. etal. (1995) FASEB J. 9:446-52; Fujimori, Y. (1993) Agents Action 39:42-8.It has also been found that inflammation in animal models of arthritiswas accompanied by activation of the contact system. Blais, C. Jr. etal. (1997) Arthritis Rheum. 40:1327-33.

Additionally, plasma kallikrein inhibitor P8720 has been found to reduceinflammation in an acute and chronic rat model of inflammatory boweldisease (IBD). Stadnicki, A. et al. (1998) FASEB J. 12:325-33;Stadnicki, A. et al. (1996) Dig. Dis. Sci. 41:912-20; and De La Cadena,R. A., et al. (1995) FASEB J 9:446-52. The contact system is activatedduring acute and chronic intestinal inflammation. Sartor, R. B. et al.(1996) Gastroenterology 110:1467-81. It has been found that B2 receptorantagonist, an antibody to high molecular weight kininogen, or reductionin levels of kininogen reduced clinicopathology in animal models of IBD.Ibid.; Arai, Y. et al. (1999) Dig. Dis. Sci. 44:845-51; and Keith, J. C.et al. (2005) Arthritis Res. Therapy 7:R769-76.

H-D-Pro-Phe-Arg-chloromethylketone (CMK), an inhibitor of PK and FXIIand a physiological inhibitor (C1-inhibitor), has been found to reducevascular permeability in multiple organs and reduce lesions inlipopolysaccharide (LPS)—or bacterial-induced sepsis in animals. Liu, D.et al. (2005) Blood 105:2350-5; Persson, K. et al. (2000)J. Exp. Med.192:1415-24. Clinical improvement was observed in sepsis patientstreated with C1-inhibitor. Zeerleder, S. et al. (2003) Clin. Diagnost.Lab. Immunol. 10:529-35; Caliezi, C., et al. (2002) Crit. Care Med.30:1722-8; and Marx, G. et al. (1999) Intensive Care Med. 25:1017-20.Fatal cases of septicemia are found to have a higher degree of contactactivation. Martinez-Brotons, F. et al. (1987) Thromb. Haemost.58:709-713; and Kalter, E. S. et al. (1985) J. Infect. Dis. 151:1019-27.

It has also been found that prePK levels are higher in diabetics,especially those with proliferative retinopathy, and correlate withfructosamine levels. Gao, B.-B., et al. (2007) Nature Med. 13:181-8; andKedzierska, K. et al. (2005) Archives Med. Res. 36:539-43. PrePK is alsofound to be highest in those with a sensorimotor neuropathy. Christie,M. et al. (1984) Thromb. Haemostas. (Stuttgart) 52:221-3. PrePK levelsare elevated in diabetics and are associated with increased bloodpressure. PrePK levels independently correlate with the albuminexcretion rate and are elevated in diabetics with macroalbuminuria,suggesting prePK may be a marker for progressive nephropathy. Jaffa, A.A. et al. (2003) Diabetes 52:1215-21. B1 receptor antagonists have beenfound to decrease plasma leakage in rats treated with streptozotocin.Lawson, S. R. et al. (2005) Eur. J. Pharmacol. 514:69-78. B1 receptorantagonists can also prevent streptozotocin-treated mice from developinghyperglycemia and renal dysfunction. Zuccollo, A. et al. (1996) Can. J.Physiol. Pharmacol. 74:586-9.

In certain aspects, the invention provides a crystalline salt ofCompound I, for use as a medicament.

In certain aspects, the invention provides methods of treating orpreventing a disease or condition characterized by aberrant plasmakallikrein activity. The method includes the step of administering to asubject in need thereof a therapeutically effective amount of acrystalline salt of Compound I, thereby treating or preventing thedisease or condition characterized by aberrant plasma kallikreinactivity. By reducing plasma kallikrein activity in the subject, thedisease or condition characterized by aberrant plasma kallikreinactivity is treated.

The terms “treat,” “treating,” and “treatment” as used herein meansprevent, halt or slow the progression of, or eliminate a disease orcondition in a subject. In some embodiments “treat,” “treating,” and“treatment” means halt or slow the progression of, or eliminate adisease or condition in a subject. In some embodiments, “treat,”“treating,” and “treatment” means reducing at least one objectivemanifestation of a disease or condition in a subject.

The term “effective amount” as used herein refers to an amount that issufficient to bring about a desired biological effect.

The term “therapeutically effective amount” as used herein refers to anamount that is sufficient to bring about a desired therapeutic effect.

The term “inhibit” as used herein means decrease by an objectivelymeasurable amount or extent. In various embodiments “inhibit” meansdecrease by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95percent compared to relevant control. In one embodiment “inhibit” meansdecrease 100 percent, i.e., halt or eliminate.

The term “subject” as used herein refers to a mammal. In variousembodiments, a subject is a mouse, rat, rabbit, cat, dog, pig, sheep,horse, cow, or non-human primate. In one embodiment, a subject is ahuman.

Alternatively, in certain aspects, the invention provides a crystallinesalt of Compound I for treatment of a disease or condition characterizedby aberrant plasma kallikrein activity.

Alternatively, in certain aspects, the invention provides the use of acrystalline salt of Compound I for the manufacture of a medicament foruse in treatment of a disease or condition characterized by aberrantplasma kallikrein activity.

As used herein, a “disease or condition characterized by aberrant plasmakallikrein activity” refers to any disease or condition in which it isdesirable to reduce plasma kallikrein activity. For example, it may bedesirable to reduce plasma kallikrein activity in the setting ofinappropriate activation or hyperactivation of kallikrein. As anotherexample, it may be desirable to reduce plasma kallikrein activity in thesetting of a hypercoagulable state. As another example, it may bedesirable to reduce plasma kallikrein activity in the setting of tissueischemia that is associated with the presence or formation of thrombus.

In certain embodiments, the disease or condition characterized byaberrant plasma kallikrein activity is selected from the groupconsisting of stroke, inflammation, reperfusion injury, acute myocardialinfarction, deep vein thrombosis, post fibrinolytic treatment condition,angina, edema, angioedema, hereditary angioedema, sepsis, arthritis,hemorrhage, blood loss during cardiopulmonary bypass, inflammatory boweldisease, diabetes mellitus, retinopathy, diabetic retinopathy, diabeticmacular edema, diabetic macular degeneration, age-related macular edema,age-related macular degeneration, proliferative retinopathy, neuropathy,hypertension, brain edema, increased albumin excretion,macroalbuminuria, and nephropathy.

In certain embodiments, the disease or condition characterized byaberrant plasma kallikrein activity is angioedema.

In certain embodiments, the disease or condition characterized byaberrant plasma kallikrein activity is acquired angioedema or hereditaryangioedema (HAE).

Acquired Angioedema (AAE) (Caldwell J R, et al. Clin ImmunolImmunopathol. 1972; 1:39-52) is characterized in several ways, includingby acquired deficiency of C1 inhibitor (C1-INH), hyperactivation of theclassical pathway of human complement and angioedema symptoms mediatedby bradykinin released by inappropriate activation of the contact-kininsystem. AAE may be present in two forms, AAE type 1 (which is normallyassociated with another disease) and AAE type II, which is normallyassociated with an autoimmune disease. AAE may be caused by a number offactors, including, but not limited to, autoimmune diseases (forexample, the production of anti-C INH antibodies) or by an acquiredmutation in C1 INH. Furthermore, the crystalline salts of Compound I maybe used to treat side effects of angiotensin converting enzyme (ACE)inhibitor treatments. ACE inhibitors block the major pathway forbreakdown of bradykinin. Inhibiting kallikrein formation through the useof the crystalline salts of the invention reduces the formation ofbradykinin.

In certain embodiments, the disease or condition characterized byaberrant plasma kallikrein activity is hereditary angioedema (HAE). Incertain embodiments, the hereditary angioedema is Type I hereditaryangioedema. Alternatively, the hereditary angioedema may be Type IIhereditary angioedema. Alternatively, the hereditary angioedema may beType III hereditary angioedema.

In certain embodiments, the crystalline salt of Compound I is used forprophylactic treatment of HAE. In other embodiments, the crystallinesalt of Compound I is used for acute treatment of HAE.

In certain embodiments, the crystalline salt of Compound I is used forthe prevention or treatment of angioedema attacks in a subject with HAE.In certain embodiments, the crystalline salt of Compound I is used as apreventive treatment to reduce the frequency of angioedema attacks in asubject with HAE. In other embodiments, the crystalline salt of CompoundI is used for the treatment of an acute angioedema attack in a subjectwith HAE.

In certain embodiments, the disease or condition characterized byaberrant plasma kallikrein activity is stroke.

In certain embodiments, the disease or condition characterized byaberrant plasma kallikrein activity is reperfusion injury.

In certain embodiments, the disease or condition characterized byaberrant plasma kallikrein activity is acute myocardial infarction.

In certain embodiments, the disease or condition characterized byaberrant plasma kallikrein activity is hemorrhage.

In certain embodiments, the disease or condition characterized byaberrant plasma kallikrein activity is blood loss during cardiopulmonarybypass.

In certain embodiments, the disease or condition characterized byaberrant plasma kallikrein activity is selected from the groupconsisting of retinopathy, diabetic retinopathy, diabetic macular edema,diabetic macular degeneration, age-related macular edema, age-relatedmacular degeneration, and proliferative retinopathy.

Formulations, Routes of Administration, and Dosing

The crystalline salts of Compound I described herein can be formulatedas pharmaceutical compositions and administered to a mammalian host,such as a human patient, in a variety of forms adapted to the chosenroute of administration, e.g., orally or parenterally, by intravenous,intraperitoneal, intramuscular, topical, or subcutaneous routes.Additional routes of administration are also contemplated by theinvention.

Thus, the crystalline salts of Compound I (also referred to herein as an“active compound”) may be systemically administered, e.g., orally, incombination with a pharmaceutically acceptable vehicle such as an inertdiluent or an assimilable edible carrier. They may be enclosed in hardor soft shell gelatin capsules, may be compressed into tablets, or maybe incorporated directly with the food of the patient's diet. For oraltherapeutic administration, the active compound may be combined with oneor more excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2% to about 60% of the weight of a given unit dosage form. Theamount of active compound in such therapeutically useful compositions issuch that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing diluents and carriers: binders such as gum tragacanth, acacia,corn starch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose or aspartame or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring may beadded. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, theactive compound may be incorporated into sustained-release preparationsand devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound can be prepared in water or physiologically acceptable aqueoussolution, optionally mixed with a nontoxic surfactant. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, triacetin,and mixtures thereof and in oils. Under ordinary conditions of storageand use, these preparations contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active compound which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, methods of preparation can includevacuum drying and the freeze drying techniques, which yield a powder ofthe active compound plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the crystalline salts of Compound I may beapplied in pure form, i.e., when they are prepared in liquids. However,it will generally be desirable to administer them to the skin ascompositions or formulations, in combination with a dermatologicallyacceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the crystalline salts of the invention can be dissolvedor dispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the crystalline salts of the invention to the skin are known inthe art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392;incorporated herein by reference), Geria (U.S. Pat. No. 4,992,478;incorporated herein by reference), Smith et al. (U.S. Pat. No.4,559,157; incorporated herein by reference), and Wortzman (U.S. Pat.No. 4,820,508; incorporated herein by reference).

Useful dosages of the crystalline salts of Compound I can be determined,at least initially, by comparing their in vitro activity and in vivoactivity in animal models. Methods for the extrapolation of effectivedosages in mice, and other animals, to humans are known in the art; forexample, see U.S. Pat. No. 4,938,949 (incorporated herein by reference).

The amount of the crystalline salt of Compound I required for use intreatment will vary not only with the particular crystalline saltselected but also with the route of administration, the nature of thecondition being treated, and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 100 mg/kg body weight of the recipient per day, e.g., fromabout 3 to about 90 mg/kg of body weight per day, from about 6 to about75 mg per kilogram of body weight per day, from about of 10 to about 60mg/kg of body weight per day, or from about 15 to about 50 mg/kg of bodyweight per day.

Crystalline salts of Compound I can be conveniently formulated in unitdosage form; for example, containing 5 to 1000 mg, 10 to 750 mg, 50 to500 mg, 75 mg to 350 mg, 75 mg to 300 mg, 75 mg to 250 mg, 75 mg to 200mg, 75 mg to 175 mg, 75 mg to 150 mg, 75 mg to 125 mg, 100 mg to 750 mg,100 mg to 500 mg, 100 mg to 350 mg, 100 mg to 300 mg, 100 mg to 250 mg,100 mg to 200 mg, 100 mg to 175 mg, 100 mg to 150 mg, 100 mg to 125 mg,125 mg to 350 mg, 125 mg to 300 mg, 125 mg to 250 mg, 125 mg to 200 mg,125 mg to 175 mg, 125 mg to 150 mg, including, for example, 5 mg, 10 mg,25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 250 mg, 300mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, and other such unit dosagesfalling within the foregoing unit dosage ranges, of active compound perunit dosage form. In one embodiment, the invention provides acomposition comprising a crystalline salt of Compound I formulated insuch a unit dosage form. The desired dose may conveniently be presentedin a single dose or as divided doses to be administered at appropriateintervals, for example, as two, three, four or more sub-doses per day.The sub-dose itself may be further divided, e.g., into a number ofdiscrete loosely spaced administrations.

Crystalline salts of Compound I can also be administered in combinationwith other therapeutic agents, for example, other agents that are usefulfor treating or preventing ischemia, blood loss, or reperfusion injury.

Other delivery systems can include time-release, delayed release, orsustained release delivery systems such as are well-known in the art.Such systems can avoid repeated administrations of the active compound,increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available and known to those of ordinaryskill in the art. Use of a long-term sustained release implant may bedesirable. Long-term release, as used herein, means that the deliverysystem or is implant constructed and arranged to deliver therapeuticlevels of the active compound for at least 30 days, and preferably 60days.

In certain embodiments, a crystalline salt of Compound I is formulatedfor intraocular administration, for example direct injection orinsertion within or in association with an intraocular medical device.

The crystalline salts of Compound I may be formulated for depositinginto a medical device, which may include any of a variety ofconventional grafts, stents, including stent grafts, catheters,balloons, baskets, or other device that can be deployed or permanentlyimplanted within a body lumen. As a particular example, it would bedesirable to have devices and methods which can deliver crystallinesalts of the invention to the region of a body which has been treated byinterventional technique.

In exemplary embodiments, a crystalline salt of Compound I may bedeposited within a medical device, such as a stent, and delivered to thetreatment site for treatment of a portion of the body.

Stents have been used as delivery vehicles for therapeutic agents (i.e.,drugs). Intravascular stents are generally permanently implanted incoronary or peripheral vessels. Stent designs include those of U.S. Pat.No. 4,733,655 (Palmaz), U.S. Pat. No. 4,800,882 (Gianturco), or U.S.Pat. No. 4,886,062 (Wiktor). Such designs include both metal andpolymeric stents, as well as self-expanding and balloon-expandablestents. Stents may also be used to deliver a drug at the site of contactwith the vasculature, as disclosed in U.S. Pat. No. 5,102,417 (Palmaz),U.S. Pat. No. 5,419,760 (Narciso, Jr.), U.S. Pat. No. 5,429,634(Narciso, Jr.), and in International Patent Application Nos. WO 91/12779(Medtronic, Inc.) and WO 90/13332 (Cedars-Sanai Medical Center), forexample.

The term “deposited” means that the active compound is coated, adsorbed,placed, or otherwise incorporated into the device by methods known inthe art. For example, the compound may be embedded and released fromwithin (“matrix type”) or surrounded by and released through (“reservoirtype”) polymer materials that coat or span the medical device. In thelatter example, the compound may be entrapped within the polymermaterials or coupled to the polymer materials using one or more thetechniques for generating such materials known in the art. In otherformulations, the compound may be linked to the surface of the medicaldevice without the need for a coating, for example by means ofdetachable bonds, and release with time or can be removed by activemechanical or chemical processes. In other formulations, the compoundmay be in a permanently immobilized form that presents the compound atthe implantation site.

In certain embodiments, the active compound may be incorporated withpolymer compositions during the formation of biocompatible coatings formedical devices, such as stents. The coatings produced from thesecomponents are typically homogeneous and are useful for coating a numberof devices designed for implantation.

The polymer may be either a biostable or a bioabsorbable polymerdepending on the desired rate of release or the desired degree ofpolymer stability, but frequently a bioabsorbable polymer is preferredfor this embodiment since, unlike a biostable polymer, it will not bepresent long after implantation to cause any adverse, chronic localresponse. Bioabsorbable polymers that could be used include, but are notlimited to, poly(L-lactic acid), polycaprolactone, polyglycolide (PGA),poly(lactide-co-glycolide) (PLLA/PGA), poly(hydroxybutyrate),poly(hydroxybutyrate-co-valerate), polydioxanone, polyorthoester,polyanhydride, poly(glycolic acid), poly(D-lactic acid), poly(L-lacticacid), poly(D, L-lactic acid), poly(D, L-lactide) (PLA), poly(L-lactide) (PLLA), poly(glycolic acid-co-trimethylene carbonate)(PGA/PTMC), polyethylene oxide (PEO), polydioxanone (PDS),polyphosphoester, polyphosphoester urethane, poly(amino acids),cyanoacrylates, poly(trimethylene carbonate), poly(iminocarbonate),copoly(ether-esters) (e.g., PEO/PLA), polyalkylene oxalates,polyphosphazenes and biomolecules such as fibrin, fibrinogen, cellulose,starch, collagen and hyaluronic acid, polyepsilon caprolactone,polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates, cross linked or amphipathic blockcopolymers of hydrogels, and other suitable bioabsorbable poplymersknown in the art. Also, biostable polymers with a relatively low chronictissue response such as polyurethanes, silicones, and polyesters couldbe used, and other polymers could also be used if they can be dissolvedand cured or polymerized on the medical device such as polyolefins,polyisobutylene and ethylene-alphaolefin copolymers; acrylic polymersand copolymers, vinyl halide polymers and copolymers, such as polyvinylchloride; polyvinylpyrrolidone; polyvinyl ethers, such as polyvinylmethyl ether; polyvinylidene halides, such as polyvinylidene fluorideand polyvinylidene chloride; polyacrylonitrile, polyvinyl ketones;polyvinyl aromatics, such as polystyrene, polyvinyl esters, such aspolyvinyl acetate; copolymers of vinyl monomers with each other andolefins, such as ethylene-methyl methacrylate copolymers,acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetatecopolymers; pyran copolymer; polyhydroxy-propyl-methacrylamide-phenol;polyhydroxyethyl-aspartamide-phenol; polyethyleneoxide-polylysinesubstituted with palmitoyl residues; polyamides, such as Nylon 66 andpolycaprolactam; alkyd resins, polycarbonates; polyoxymethylenes;polyimides; polyethers; epoxy resins, polyurethanes; rayon;rayon-triacetate; cellulose, cellulose acetate, cellulose butyrate;cellulose acetate butyrate; cellophane; cellulose nitrate; cellulosepropionate; cellulose ethers; and carboxymethyl cellulose.

Polymers and semipermeable polymer matrices may be formed into shapedarticles, such as valves, stents, tubing, prostheses and the like.

In certain embodiments of the invention, the crystalline salt ofCompound I is coupled to a polymer or semipermeable polymer matrix thatis formed as a stent or stent-graft device.

Typically, polymers are applied to the surface of an implantable deviceby spin coating, dipping, or spraying. Additional methods known in theart can also be utilized for this purpose. Methods of spraying includetraditional methods as well as microdeposition techniques with an inkjettype of dispenser. Additionally, a polymer can be deposited on animplantable device using photo-patterning to place the polymer on onlyspecific portions of the device. This coating of the device provides auniform layer around the device which allows for improved diffusion ofvarious analytes through the device coating.

In certain embodiments of the invention, the active compound isformulated for release from the polymer coating into the environment inwhich the medical device is placed. Preferably, the compound is releasedin a controlled manner over an extended time frame (e.g., months) usingat least one of several well-known techniques involving polymer carriersor layers to control elution. Some of these techniques are described inU.S. Patent Application 2004/0243225A1, the entire disclosure of whichis incorporated herein in its entirety.

Moreover, as described for example in U.S. Pat. No. 6,770,729, which isincorporated herein in its entirety, the reagents and reactionconditions of the polymer compositions can be manipulated so that therelease of the active compound from the polymer coating can becontrolled. For example, the diffusion coefficient of the one or morepolymer coatings can be modulated to control the release of the compoundfrom the polymer coating. In a variation on this theme, the diffusioncoefficient of the one or more polymer coatings can be controlled tomodulate the ability of an analyte that is present in the environment inwhich the medical device is placed (e.g. an analyte that facilitates thebreakdown or hydrolysis of some portion of the polymer) to access one ormore components within the polymer composition (and for example, therebymodulate the release of the compound from the polymer coating). Yetanother embodiment of the invention includes a device having a pluralityof polymer coatings, each having a plurality of diffusion coefficients.In such embodiments of the invention, the release of the active compoundfrom the polymer coating can be modulated by the plurality of polymercoatings.

In yet another embodiment of the invention, the release of the activecompound from the polymer coating is controlled by modulating one ormore of the properties of the polymer composition, such as the presenceof one or more endogenous or exogenous compounds, or alternatively, thepH of the polymer composition. For example, certain polymer compositionscan be designed to release a compound in response to a decrease in thepH of the polymer composition.

Kits

The invention also provides a kit, comprising a crystalline salt ofCompound I, at least one other therapeutic agent, packaging material,and instructions for administering the crystalline salt of Compound Iand the other therapeutic agent or agents to a mammal to treat orprevent a disease or condition characterized by aberrant kallikreinactivity in the mammal.

In one embodiment, the mammal is a human.

EXAMPLES

Materials and Methods

X-Ray Powder Diffraction

The XRPD pattern was collected with a PANalytical X′Pert PRO MPDdiffractometer using an incident beam of Cu radiation produced using anOptix long, fine-focus source. An elliptically graded multilayer mirrorwas used to focus Cu Kα X-rays through the specimen and onto thedetector. Prior to the analysis, a silicon specimen (NIST SRM 640e) wasanalyzed to verify the observed position of the Si 111 peak isconsistent with the NIST-certified position. A specimen of the samplewas sandwiched between 3-μm-thick films and analyzed in transmissiongeometry. A beam-stop, short antiscatter extension, and antiscatterknife edge were used to minimize the background generated by air. Sollerslits for the incident and diffracted beams were used to minimizebroadening from axial divergence. Diffraction patterns were collectedusing a scanning position-sensitive detector (X′Celerator) located 240mm from the specimen and Data Collector software v. 2.2b.

Thermogravimetric Analysis

TG analyses were performed using a TA Instruments Discoverythermogravimetric analyzer with an IR furnace. Temperature calibrationwas performed using nickel and Alumel™. Each sample was placed in analuminum pan. The sample was hermetically sealed, the lid pierced, theninserted into the TG furnace. The furnace was heated under nitrogen. Theacquisition scan rate is recorded in the thermogram header, while theheating range can be determined from the individual plot.

Infrared (IR) Spectroscopy

Thermogravimetric infrared (TG-IR) analysis was performed on a TAInstruments Q5000 IR thermogravimetric (TG) analyzer interfaced to aMagna-IR 560® Fourier transform infrared (FT-IR) spectrophotometer(Thermo Nicolet) equipped with an Ever-Glo mid/far IR source, apotassium bromide (KBr) beamsplitter, and a mercury cadmium telluride(MCT-A) detector. The FT-IR wavelength verification was performed usingpolystyrene, and the TG calibration standards were nickel and Alumel™.The sample was placed in a platinum sample pan, and the pan was insertedinto the TG furnace. The TG instrument was started first, immediatelyfollowed by the FT-IR instrument. The TG instrument was operated under aflow of helium at 90 and 10 cc/min for the purge and balance,respectively. The furnace was heated under helium at a rate of 20°C./minute to a final temperature of 350° C. IR spectra were collectedapproximately every 16 seconds for approximately 13 minutes. Each IRspectrum represents 16 co-added scans collected at a spectral resolutionof 4 cm⁻¹. Volatiles were identified from a search of the HighResolution Nicolet Vapor Phase spectral library.

Example 1: Synthetic Protocol for Racemic Compound 54e

Reproduced from WO 2015/134998 and U.S. Patent Application PublicationNo. 2017/0073314 A1 (both incorporated by reference)

Preparation of1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide(54e) Step-1: Preparation of3-((3-amino-4-fluorophenyl)(hydroxy)methyl)benzonitrile (54b)

To a solution of 3-formylbenzonitrile (54a) (29 g, 217 mmol) intetrahydrofuran (200 mL) cooled to 0° C. was added freshly preparedGrignard reagent (52c) (245 mL, 221 mmol, ˜0.9 M in THF) stirred at 0°C. for 1 h, and room temperature for 18 h. The reaction mixture wasquenched with 1 N HCl (aq. 440 mL), stirred for 3 h, neutralized withNaOH (2 N, aq.) to pH=˜8. The reaction mixture was extracted with ethylacetate (600, 300 mL). The combined extracts were washed with brine (120mL), dried over MgSO₄, filtered and concentrated in vacuum. The crudeproduct was purified by flash column chromatography [silica gel, elutingwith hexanes/ethyl acetate (1:0 to 1:1) to give3-((3-amino-4-fluorophenyl)(hydroxy)methyl)benzonitrile (54b) (36.28 g)as a brown gum which was used as such for next step; MS (ES+) 265.3(M+23).

Step-2: Preparation of tert-butyl3-(5-(5-((3-cyanophenyl)(hydroxy)methyl)-2-fluorophenylcarbamoyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzylcarbamate(54c)

To a solution of 3-((3-amino-4-fluorophenyl)(hydroxy)methyl)benzonitrile(54b) (24.682 g, 102 mmol) in DMF (480 mL) was added1-(3-((tert-butoxycarbonylamino)methyl)phenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxylicacid (10d) (35.0 g, 91 mmol), N-ethyl-N-isopropylpropan-2-amine (132 mL,758 mmol), bromotripyrrolidin-1-ylphosphonium hexafluorophosphate(V)(PyBrOP, 42.8 g, 91 mmol) and stirred at room temperature for 19 h. Thereaction mixture was diluted with ethyl acetate (1000 mL), washed withwater (500, 400 mL), brine (400 mL), dried over MgSO₄, filtered andconcentrated in vacuum. The crude product was purified by flash columnchromatography [silica gel, eluting with hexanes/ethyl acetate (1:0 to1:1)] to afford tert-butyl3-(5-(5-((3-cyanophenyl)(hydroxy)methyl)-2-fluorophenylcarbamoyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzylcarbamate(54c) (4.583 g, 5% for two steps) as a yellow solid; ¹H NMR (300 MHz,DMSO-d₆) δ 10.57 (s, 1H), 7.81 (t, J=1.7 Hz, 1H), 7.73-7.66 (m, 2H),7.64-7.19 (m, 10H), 6.25 (d, J=4.0 Hz, 1H), 5.78 (d, J=4.0 Hz, 1H), 4.19(d, J=6.1 Hz, 2H), 1.37 (s, 9H); ¹⁹F NMR (282 MHz, DMSO-d₆) δ-60.81,−123.09; MS (ES+) 632.3 (M+23).

Step-3: Preparation of tert-butyl3-(5-(5-((3-cyanophenyl)(cyclopropylmethylamino)methyl)-2-fluorophenylcarbamoyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzylcarbamate(54d)

To a solution of tert-butyl3-(5-(5-((3-cyanophenyl)(hydroxy)methyl)-2-fluorophenylcarbamoyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzylcarbamate(54c) (1.333 g, 2.187 mmol) in dichloromethane (40 mL) at 0° C. wasadded thionyl chloride (0.340 mL, 4.59 mmol) and warmed to roomtemperature over 2 h. The reaction mixture was quenched with triethylamine (2.0 mL, 14.35 mmol) stirred at room temperature for 1 h. It wasthen treated with cyclopropylmethanamine (4.30 mL, 48.0 mmol),concentrated to remove most of dichloromethane followed by addition ofacetonitrile (30 mL), stirring at 70° C. for 14 h, and concentration invacuum to dryness. The residue was treated with chloroform (200 mL),washed with water (100 mL), dried over MgSO₄ followed by filtration andconcentration. The crude product was purified by flash columnchromatography [silica gel eluting with hexanes/ethyl acetate (1:0 to2:1)] to afford tert-butyl3-(5-(5-((3-cyanophenyl)(cyclopropylmethylamino)methyl)-2-fluorophenylcarbamoyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzylcarbamate(54d) (184 mg, 13%) as colorless gum; ¹H NMR (300 MHz, DMSO-d₆) δ 10.56(s, 1H), 7.89 (t, J=1.7 Hz, 1H), 7.77-7.71 (m, 1H), 7.70-7.30 (m, 10H),7.22 (dd, J=10.3, 8.5 Hz, 1H), 4.93 (s, 1H), 4.19 (d, J=6.2 Hz, 2H),2.26 (d, J=6.6 Hz, 2H), 1.37 (s, 9H), 1.00-0.80 (m, 1H), 0.45-0.28 (m,2H), 0.12-−0.01 (m, 2H); ¹⁹F NMR (282 MHz, DMSO-d₆) δ-60.80, −123.20; MS(ES+) 663.4 (M+1).

Step-4: Preparation of1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide(54e)

To a solution of tert-butyl3-(5-(5-((3-cyanophenyl)(cyclopropylmethylamino)methyl)-2-fluorophenylcarbamoyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzylcarbamate(54d) (161 mg, 0.243 mmol) in 1,4-Dioxane (18 mL) was added hydrogenchloride (2.60 mL, 10.40 mmol, 4 M in 1,4-dioxane) and stirred at roomtemperature for 16 h. the reaction mixture was treated with hexanes,decanted, washed with hexanes, and decanted again. The insoluble crudeproduct was purified by flash column chromatography [silica gel, elutingwith chloroform/CMA80 (1:0 to 2:1)] to afford1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide(54e). The pure product was dissolved in methanol (10 mL) and added 4 NHCl (aq. 0.14 mL) followed by concentration in vacuum to dryness to giveHCl salt of1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide(54e) (74 mg, 48%) white solid; ¹H NMR (300 MHz, DMSO-d₆, D2O ex NMR) δ8.13 (t, J=1.7 Hz, 1H), 7.98-7.84 (m, 3H), 7.73-7.64 (m, 3H), 7.63-7.48(m, 4H), 7.44 (dd, J=10.2, 8.6 Hz, 1H), 5.75 (s, 1H), 4.12 (s, 2H), 2.76(d, J=7.2 Hz, 2H), 1.17-0.94 (m, 1H), 0.68-0.47 (m, 2H), 0.34-0.24 (m,2H); ¹⁹F NMR (282 MHz, DMSO-d₆) δ-60.82, −120.02; MS (ES+): 563.3 (M+1);Analysis calculated for C₃₀H₂₆F₄N₆O·2.0 HCl·3.0 H₂O: C, 52.26; H, 4.97;N, 12.19; Found: C, 52.26; H, 5.00; N, 11.72.

Example 2: Separation of Enantiomers of Racemic Compound 54e

Reproduced from WO 2015/134998 and U.S. Patent Application PublicationNo. 2017/0073314 A1 (both incorporated by reference)

Separation of(+)-1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide(Compound I), and(−)-1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide((−)-enantiomer)

Isomers of Racemic1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide(54e) (0.4 g) were separated by using preparative SFC method using thefollowing conditions to furnish:

Preparative SFC Method used: Column 20 mm × 25.0 cm ChromegaChiral CCSfrom Regis Technologies (Morton Grove, IL) CO₂ Co-solvent (Solvent B)Methanol:Isopropanol (1:1) with 1% Isopropylamine Isocratic Method 20%Co-solvent at 80 mL/min System Pressure 200 bar Column Temperature 25°C. Sample Diluent Methanol: Isopropanol Chiral Purity of peaks wasdetermined by following Analytical SFC Method: Column 4.6 × 100 mmChiralPak AS from Chiral Technologies (West Chester, PA) CO₂ Co-solvent(Solvent B) Methanol:Isopropanol (1:1) with 0.1% IsopropylamineIsocratic Method 5-65% Co-solvent Gradient at 4 mL/min System Pressure100 bar Column Temperature 25° C. Sample Diluent Methanol Peak-1(Compound I) 2.1 min 144 mg >95% ee (UV 254) 98.6% purity (UV 254)Peak-2 ((−)-enantiomer) 2.4 min 172 mg 95.5% ee (UV 254) 96.5% purity(UV 254)

-   1. Peak-1 assigned as    (+)-1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide    (Compound I) (144 mg, >95% ee) free base as white solid; Optical    rotation: [α]_(D)=(+) 6.83 [CH₃OH, 1.2]; ¹H NMR (300 MHz, DMSO-d₆) δ    10.53 (s, 1H, D₂O exchangeable), 7.88 (t, J=1.7 Hz, 1H), 7.77-7.71    (m, 1H), 7.67 (dt, J=7.7, 1.4 Hz, 1H), 7.63 (dd, J=7.5, 2.1 Hz, 1H),    7.56 (s, 1H), 7.54-7.47 (m, 2H), 7.47-7.38 (m, 2H), 7.34 (ddt,    J=8.6, 5.9, 2.8 Hz, 2H), 7.22 (dd, J=10.3, 8.5 Hz, 1H), 4.93 (s,    1H), 3.77 (s, 2H), 2.31-2.21 (m, 2H), 0.97-0.80 (m, 1H), 0.42-0.33    (m, 2H), 0.10-−0.02 (m, 2H); ¹⁹F NMR (282 MHz, DMSO-d₆) δ-60.73,    −123.20; MS (ES+) 563.3 (M+1), 561.3 (M−1). To a solution of free    base mixture of    (+)-1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide    (Compound I) (120 mg) in methanol (15 mL) was added hydrogen    chloride (0.969 mL, 1.938 mmol), stirred at room temperature for 10    min, evaporated to dryness to afford    (+)-1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide    (Compound I) (100 mg) hydrochloride salt as white solid; ¹H NMR (300    MHz, DMSO-d₆) δ 10.84 (s, 1H, D₂O exchangeable), 10.44 (s, 2H, D₂O    exchangeable), 8.44 (s, 3H, D₂O exchangeable), 8.30 (s, 1H, D₂O    exchangeable), 8.09 (d, J=7.9 Hz, 1H), 7.99 (d, J=6.8 Hz, 1H),    7.91-7.83 (m, 1H), 7.80-7.50 (m, 7H), 7.42 (dd, J=10.3, 8.6 Hz, 1H),    5.78 (d, J=6.9 Hz, 1H), 4.13 (d, J=5.7 Hz, 2H), 2.88-2.62 (m, 2H),    1.42-0.99 (m, 1H), 0.73-0.46 (m, 2H), 0.32 (d, J=4.4 Hz, 2H); ¹⁹F    NMR (282 MHz, DMSO-d₆) 5-60.81, −119.99; MS (ES+): MS (ES+) 563.3    (M+1), MS (ES−) 561.3 (M−1), 597.3 (M+Cl); Analysis calculated for    C₃₀H₂₆F₄N₆O·2HCl·1.75H₂O: C, 54.02; H, 4.76; Cl, 10.63; N, 12.60;    Found: C, 54.12; H, 4.83; Cl, 10.10; N, 11.97.-   2. Peak-2 assigned as    (−)-1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide    ((−)-enantiomer) (172 mg, 95.5% ee) as free base was repurified by    flash column chromatography (silica gel 12 g, eluting 0-30% MeOH in    chloroform for 15 min) to afford    (−)-1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide    ((−)-enantiomer) free base as an off-white solid; Optical rotation:    [α]_(D)=(−) 5.44 [CH₃OH, 1.25]; ¹H NMR (300 MHz, DMSO-d₆) δ 7.88 (t,    J=1.6 Hz, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.70-7.61 (m, 2H), 7.57 (s,    1H), 7.54-7.47 (m, 2H), 7.45-7.41 (m, 2H), 7.34 (ddq, J=8.7, 6.1,    3.5, 2.8 Hz, 2H), 7.22 (dd, J=10.3, 8.5 Hz, 1H), 4.93 (s, 1H), 3.78    (s, 2H), 2.25 (d, J=6.9 Hz, 2H), 0.90 (ddd, J=9.8, 8.0, 5.2 Hz, 1H),    0.47-0.29 (m, 2H), 0.04 (dd, J=5.0, 1.5 Hz, 2H); ¹⁹F NMR (282 MHz,    DMSO-d₆) δ-60.73, −123.19; MS (ES+) 563.3 (M+1), MS (ES−), 561.3    (M−1). To a solution of free base of    (−)-1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide    ((−)-enantiomer) (0.124 g, 0.220 mmol) in methanol (15 mL) was added    hydrogen chloride (1.102 mL, 2.204 mmol), stirred at room    temperature for 10 min, evaporated to dryness to afford    (−)-1-(3-(aminomethyl)phenyl)-N-(5-((3-cyanophenyl)(cyclopropyl-methylamino)methyl)-2-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide    ((−)-enantiomer) (0.121 g) hydrochloride salt as an off-white solid;    ¹H NMR: ¹H NMR (300 MHz, DMSO-d₆) δ 10.82 (s, 1H, D₂O exchangeable),    10.36 (s, 2H, D₂O exchangeable), 8.38 (s, 3H, D₂O exchangeable),    8.27 (s, 1H), 8.06 (d, J=7.9 Hz, 1H), 7.98 (d, J=6.7 Hz, 1H), 7.87    (d, J=7.7 Hz, 1H), 7.78-7.49 (m, 7H), 7.48-7.37 (m, 1H), 5.78 (s,    1H), 4.13 (d, J=5.7 Hz, 2H), 2.72 (s, 2H), 1.14 (s, 1H), 0.56 (d,    J=7.7 Hz, 2H), 0.31 (d, J=5.0 Hz, 2H); ¹⁹F NMR (282 MHz, DMSO-d₆)    δ-60.82, −120.03; MS (ES+): MS (ES+) 563.3 (M+1), MS (ES−), 561.3    (M−1), 597.2 (M+Cl); Analysis calculated for    C₃₀H₂₆F₄N₆O·2HCl·1.75H₂O: C, 54.02; H, 4.76; Cl, 10.63; N, 12.60;    Found: C, 54.12; H, 4.83; Cl, 10.10; N, 11.97.

Example 3: Preparation of a Seed Crystal of Compound I•2(HCl)

A solution of Compound I (see Example 2) in methyl tert-butyl ether(MTBE) (1 equiv) is added to a solution of HCl (aq) (2 equiv) inmethanol (cold), followed by heating to about 30° C., and keeping it atabout 30° C. for not longer than 5 hours while stirring at about 115rpm. Compound I bis(HCl) is collected by filtration and dried. Thecrystalline material obtained can be used as a seed for thecrystallization protocol described in Example 4.

Example 4: Large-Scale Synthetic & Crystallization Protocol for CompoundI•2(HCl)

37% Aqueous hydrochloric acid (38.1 kg, 32.3 L, 2.14 equiv.) was chargedto a clean and empty crystallization vessel, methanol (228.9 kg, 39.5equiv.) was added, and the contents were cooled to −7±3° C. A solutionof Compound I free base (approx. 101.8 kg; 180.9 moles) in MTBE (approx.1,300 L) was filtered through a polish filter into the crystallizationvessel at temperature −5±5° C. After rinse with MTBE, pre-weighedCompound I•2(HCl) seed crystals (1.39 kg, 0.012 equiv.; Example 3) werecharged to the crystallization vessel via the manhole. The vesselcontent was heated to 30-33° C., and the agitation speed was set to25-50 rpm. After confirmed crystallization, the slurry was agitated foranother three to four hours. The product slurry was transferred tocentrifuge and isolated by centrifugation. The product was washed withMTBE (585 L). After dry spinning the wet product, Compound I•2(HCl), itwas discharged from the centrifuge, and the product was dried at <40° C.under vacuum in a cone drier. Product Compound I•2(HCl) yield: 100 kg;157.4 mol; approx. 85%.

¹H NMR (300 MHz, DMSO-d₆) data is shown in the following table:

Chemical Number of Structure Shift (ppm) Class Hydrogens

0.02-0.10 0.33-0.42 0.80-0.97 2.21-2.31 3.77 4.93 7.22 7.34 7.38-7.477.47-7.54 7.56 7.63 7.67 7.71-7.77 7.88 10.53  m m m m s s dd ddt m m sdd dt m t s 2 2 1 2 2 1 1 2 2 2 1 1 1 1 1 1

¹⁹F NMR (282 MHz, DMSO-d₆) data is shown in the following table:

Fluorine Chemical Structure Shifts (ppm)

−60.81, −119.99

Compound I has two basic sites. The conjugate acid of the primary aminewas calculated to have a pKa value of 8.89, and the conjugate acid ofthe secondary amine was calculated to have a pKa value of 7.86.

The XRPD pattern of Compound I•2(HCl) is shown in FIG. 1. CompoundI•2(HCl) has characteristic peaks in its XRPD pattern at values of twotheta (°2θ) of 5.28, 8.96, 14.27, 16.18, 19.79, 21.16, 22.01, 23.31,24.64, and 30.31.

TG-IR analysis indicated two, distinct weight loss regions: the firstwas completed by 125° C. while the second began at approximately 208° C.IR analysis of the off gasses from this experiment detected only traceamounts of water at the initial weight loss while HCl gas was detectedat the 208° C. event. No other solvents were detected in the sample.Thus, it was determined that Compound I•2(HCl) initially loses waterwhen heated and, when heated to above 200° C., the salt begins to breakapart and HCl gas is evolved. The IR signal for all these events is veryweak indicating that they are occurring over a range and not at aspecified temperature. An exemplary TG-IR spectrum is shown in FIG. 2.

Example 5: Compound Assays

Compound I was assayed in an in vitro biochemical assay measuringinhibition of human plasma kallikrein activity. Experimental protocolsand results of the assays are found in WO 2015/134998 and U.S. PatentApplication Publication No. 2017/0073314 A1 (both incorporated byreference). Results of this biochemical assay demonstrate that CompoundI is a potent inhibitor of human plasma kallikrein activity.

Example 6: Pharmacokinetics of Compound I•2(HCl) in Healthy Subjects

As part of a Phase I, double-blind, placebo-controlled dose-rangingstudy, the pharmacokinetics of multiple ascending oral doses of CompoundI•2(HCl) were evaluated in healthy subjects. Four ascending dose cohortswere enrolled for dosing in a sequential manner. At the start, twelvesubjects were randomized into each cohort for administration of a 7-dayor 14-day course of study drug (n=10/cohort received Compound I•2(HCl)and n=2/cohort received matching placebo) according to the followingdose regimens: 125 mg Compound I•2(HCl) or placebo once per day (QD) for7 days in Cohort 1; 250 mg Compound I•2(HCl) or placebo QD for 7 days inCohort 2; 500 mg Compound I•2(HCl) or placebo QD for 7 days in Cohort 3;or 350 mg Compound I•2(HCl) or placebo QD for 14 days in Cohort 4.Overall, 37 out of the 40 subjects receiving multiple doses of CompoundI•2(HCl) completed their respective dose regimens and were available forassessment of PK parameters at either Day 7 or Day 14.

Serial blood samples were drawn for pharmacokinetic analysis before doseadministration on Day 7 or Day 14, and over the following 24 hours. Theplasma samples for determination of Compound I•2(HCl) concentrationswere analyzed with the use of a validated LC-MS/MS (LiquidChromatography with tandem mass spectrometry) bioanalytical assay.

Plasma Compound I•2(HCl) concentration versus time profiles on Day 7after QD oral administration of Compound I•2(HCl) at 125, 250, and 500mg QD, and on Day 14 after QD oral administration of Compound I•2(HCl)at 350 mg QD, are shown in FIG. 3.

Table 1 provides a summary of plasma pharmacokinetic parameters [themaximum (peak) plasma drug concentration (C_(max)), time (observed timepoint) to reach maximum (peak) plasma drug concentration following drugadministration (T_(max)), the observed trough plasma concentration atthe end of the dosing interval (Ctau), and the area under the plasmaconcentration-time curve over the dosing interval (AUC_(tau))] following7 or 14 days of multiple oral doses of Compound I•2(HCl).

TABLE 1 Summary of plasma pharmacokinetic parameters following 7 or 14days of multiple oral doses of Compound I•2(HCl) in healthy subjects(Day 7 or Day 14). Compound I•2(HCl) 125 mg QD 250 mg QD 500 mg QD 350mg QD Pharmacokinetic Day 7 Day 7 Day 7 Day 14 Parameter (N = 10) (N =9) (N = 9) (N = 9) C_(max) (ng/mL)^(a) 97.8 (35) 217 (25) 517 (37) 363(37) T_(max) (hr)^(b)      5.0 (2.0, 8.1)      6.0 (3.0, 12.0)     2.0(1.0, 8.1)     4.0 (1.0, 8.0) C_(tau) (ng/mL)^(a) 46.1 (46) 101 (25) 235(32) 158 (40) AUC_(tau) 1600 (41)  3710 (23)  8230 (34)  5720 (34)  (ng· hr/mL)^(a) ^(a)Data reported as geometric mean (CV % of geometricmean). ^(b)T_(max) reported as median (min, max).

Maximal plasma Compound I•2(HCl) concentrations were achieved atapproximately 2 to 6 hours post-dose. An increase in dosage from 125 mgto 250 mg QD and 250 mg to 500 mg QD imparted reasonable doseproportionality. Across the full 4-fold dose range, exposure increasedin a slightly greater than dose proportional manner with a 5.1- and5.3-fold increase in geometric mean values of AUC_(tau) and C_(max),respectively.

INCORPORATION BY REFERENCE

All U.S. patents and U.S. and PCT published patent applicationsmentioned herein are hereby incorporated by reference in their entiretyas if each individual patent or published application was specificallyand individually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

We claim:
 1. A pharmaceutical composition, comprising a crystalline saltof Compound I,

and a pharmaceutically acceptable carrier; wherein the pharmaceuticalcomposition is an oral dosage form; and the oral dosage form containsabout 75 mg to about 250 mg of the crystalline salt of Compound I. 2.The pharmaceutical composition of claim 1, wherein the crystalline saltof Compound I is a hydrochloride salt.
 3. The pharmaceutical compositionof claim 1, wherein the crystalline salt of Compound I is abis(hydrochloride) salt.
 4. The pharmaceutical composition of claim 3,wherein the oral dosage form contains about 75 mg to about 175 mg of thecrystalline salt of Compound I.
 5. The pharmaceutical composition ofclaim 3, wherein the oral dosage form contains about 100 mg to about 250mg of the crystalline salt of Compound I.
 6. The pharmaceuticalcomposition of claim 3, wherein the oral dosage form contains about 100mg to about 200 mg of the crystalline salt of Compound I.
 7. Thepharmaceutical composition of claim 3, wherein the oral dosage formcontains about 100 mg to about 175 mg of the crystalline salt ofCompound I.
 8. The pharmaceutical composition of claim 3, wherein theoral dosage form contains about 125 mg to about 250 mg of thecrystalline salt of Compound I.
 9. The pharmaceutical composition ofclaim 3, wherein the oral dosage form contains about 125 mg to about 200mg of the crystalline salt of Compound I.
 10. The pharmaceuticalcomposition of claim 3, wherein the oral dosage form contains about 125mg to about 175 mg of the crystalline salt of Compound I.
 11. Thepharmaceutical composition of claim 3, wherein the oral dosage formcontains about 150 mg of the crystalline salt of Compound I.
 12. Thepharmaceutical composition of claim 3, wherein the oral dosage form is acapsule, an ingestible tablet, a buccal tablet, a troche, an elixir, asuspension, a syrup, a powder, or a wafer.
 13. The pharmaceuticalcomposition of claim 3, wherein the oral dosage form is a capsule.
 14. Amethod of treating a disease or condition characterized by aberrantplasma kallikrein activity, comprising administering to a subject inneed thereof the pharmaceutical composition of claim
 1. 15. The methodof claim 14, wherein the disease or condition characterized by aberrantplasma kallikrein activity is selected from the group consisting ofstroke, inflammation, reperfusion injury, acute myocardial infarction,deep vein thrombosis, post fibrinolytic treatment condition, angina,edema, angioedema, hereditary angioedema, sepsis, arthritis, hemorrhage,blood loss during cardiopulmonary bypass, inflammatory bowel disease,diabetes mellitus, retinopathy, diabetic retinopathy, diabetic macularedema, diabetic macular degeneration, age-related macular edema,age-related macular degeneration, proliferative retinopathy, neuropathy,hypertension, brain edema, increased albumin excretion,macroalbuminuria, and nephropathy.
 16. The method of claim 15, whereinthe disease or condition characterized by aberrant plasma kallikreinactivity is angioedema.
 17. The method of claim 15, wherein the diseaseor condition characterized by aberrant plasma kallikrein activity ishereditary angioedema.
 18. A method of preventing or treating angioedemaattacks in a subject with hereditary angioedema, comprisingadministering to a subject in need thereof the pharmaceuticalcomposition of claim
 1. 19. A method of reducing the frequency ofangioedema attacks in a subject with hereditary angioedema, comprisingadministering to a subject in need thereof the pharmaceuticalcomposition of claim
 1. 20. A method of treating an acute angioedemaattack in a subject with hereditary angioedema, comprising administeringto a subject in need thereof the pharmaceutical composition of claim 1.